The present invention generally relates to diagnostic coagulation assays, and specifically, to control samples suitable for use in connection therewith. The invention particularly relates to coagulation controls suitable for both prothrombin time and activated partial thromboplastin time assays.
Coagulation control materials are used in the clinical laboratory for quality control of the prothrombin time (PT) and activated partial thromboplastin time (APTT) assays. PT assays employ thromboplastin reagents and have been used extensively for evaluating blood coagulation associated with the extrinsic pathway. APTT assays employ an intrinsic pathway activator, such as micronized silica, and a phospholipid component of a thromboplastin reagent (without tissue factor protein) for evaluating coagulation associated with the intrinsic pathway. Both PT and APTT assays are used clinically for screening patients"" plasma for coagulation factor deficiencies. Clinical screenings are employed, for example, during routine checkups and prior to surgery. PT and APTT assays are also used for monitoring treatment with anticoagulants. For example, PT assays are routinely employed to monitor oral anti-coagulant treatment with coumarin (Warfarin(trademark), Coumadin(trademark)), and APTT assays are typically used for monitoring anticoagulant treatment with heparin.
Coagulation controls are used for quality control evaluations of the PT and APTT assay systems. The controls are essential in view of potential variation in reagents employed in these assays, potential inaccuracies in the devices used to measure clotting time, and potential effects of inaccurate anticoagulant dosage. Commercial coagulation controls have been designed to mimic three physiologic conditions: (1) xe2x80x9cControl Level Ixe2x80x9d controls mimic normal coagulation and are intended to be representative of an individual without coagulation deficiencies; (2) xe2x80x9cControl Level IIxe2x80x9d controls are intended to mimic the coagulation of an individual undergoing mild anticoagulant therapy; and (3) xe2x80x9cControl Level IIIxe2x80x9d controls are intended to mimic the coagulation of an individual undergoing relatively high anticoagulant therapy.
Various types of coagulation controls are known in the art. Typically, coagulation controls are designed to be suitable for use with (1) both the PT and APTT assays, (2) the PT assay only or (3) the APTT assay only. The controls designed exclusively for use in evaluating only the APTT assay are referred to in the art as heparin controls. While effective for APTT assays, such controls are ineffective for use in evaluating PT assay systems. The present invention is directed to general coagulation controls that can be employed for evaluating both PT and APTT assay systems.
A primary performance criteria for a coagulation control is its stability over time. Zucker et al. reported that coagulation controls prepared by buffering plasma specimens with N-2-hydroxyethylpiperazine-Nxe2x80x2-2-ethanesulfonic acid (HEPES) and lyophilizing provided stability in the reconstituted control for eight hours at 25xc2x0 C. Zucker et al., Preparation of Quality Control Specimens for Coagulation, Am. J. Clin. Path. 53:924-927 (1970). Brozovic and co-workers prepared controls from plasma samples of patients on oral anti-coagulant therapy in combination with HEPES, trisodium citrate and aprotinin, and reported stability of 4 hours to 6 hours after reconstitution when stored at 4xc2x0 C. Brozovic et al., Stability of Freeze-Dried Plasma Prepared from Patients on Oral Anticoagulants, J. Clin. Path., 26:857-863 (1973). U.S. Pat. No. 5,721,140 to Speck et al. discloses a coagulation control comprising normal human plasma, clotting factor-deficient non-primate mammalian plasma and aprotinin, and report that the controls are stable in the absence of a buffer for up to five days. Numerous other patents and literature references describe various coagulation controls. Exemplary patents include U.S. Pat. No. 3,947,378 to Babson, U.S. Pat. No. 4,007,008 to Becker et al., U.S. Pat. No. 4,056,484 to Heimburger et al. and U.S. Pat. No. 4,127,502 to Li Mutti et al.
While many variations in coagulation control compositions have been reported in the art, such controls remain limited with respect to stabilityxe2x80x94particularly once reconstituted from the lyophilized form in which they are typically sold. Reconstituted commercially-available coagulation controls stored for more than about eight hours do not provide consistent, reproducible clotting times as determined by PT and/or APTT assays. Moreover, precipitates and fibrin have been observed in such reconstituted controls. There remains a need, therefore, for coagulation controls suitable for use in connection with PT and APTT assays that have enhanced stability.
It is therefore an object of the present invention to prepare coagulation controls having enhanced stability over timexe2x80x94as demonstrated by consistent clotting times in PT and APTT assays and by the absence of formation of particulate and/or fibrin matter. It is also an object of the invention to be able to produce such controls, on a commercial scale, using readily available starting materials, conventional processing equipment, and existing U.S. Food and Drug Administration (FDA) good manufacturing practices (GMP).
Briefly, therefore, the present invention is directed to coagulation controls suitable for use in connection with both prothrombin time (PT) and activated partial thromboplastin time (APTT) assays. The coagulation control comprises plasma and an anticoagulant having activity for enhancing the activity of antithrombin III (ATIII) and/or of heparin co-factor II (HCII) against thrombin and/or against blood clotting factors such as factors IXa, Xa and/or XIa. The plasma preferably includes primate plasma and a non-primate mammalian plasma. The anticoagulant is preferably a glycosaminoglycan such as heparin.
The invention is directed, moreover, to a coagulation control comprising primate plasma, non-primate mammalian plasma, and an anticoagulant such as a glycosaminoglycan having activity as described above, with the concentration of anticoagulant ranging from about 0.01 U/ml to about 0.15 U/ml, and preferably from about 0.01 U/ml to about 0.1 U/ml, where U is heparin-equivalent units. The human plasma can be normal human plasma or an abnormal human plasma such as an activated plasma or a factor-deficient plasma. The non-primate mammalian plasma is preferably bovine plasma. The glycosaminoglycan is preferably heparin, a heparin derivative, or a heparin analog. A preferred Level I coagulation control comprises normal human plasma, bovine plasma, heparin or a heparin derivative and, optionally, activated plasma, and has a prothrombin time ranging from about 11 seconds to about 13 seconds. A preferred Level II coagulation control comprises factor-deficient human plasma, normal human plasma, bovine plasma, and heparin or a heparin derivative, and has a prothrombin time ranging from about 17 seconds to about 22 seconds. A preferred Level III coagulation control comprises factor-deficient human plasma, normal human plasma, bovine plasma, and heparin or a heparin derivative, and has a prothrombin time ranging from about 25 seconds to about 33 seconds. Each of the aforementioned prothrombin times are determined using a thromboplastin reagent having an International Sensitivity Index (ISI) value of about 2.
The invention is also directed to a coagulation control, typically in lyophilized form, that is suitable upon reconstitution for use in connection with prothrombin time (PT) and activated partial thromboplastin time (APTT) assays. The lyophilized coagulation control comprises primate plasma, non-primate mammalian plasma, and an anticoagulant such as a glycosaminoglycan having activity as described above and present in the composition at a concentration ranging from about 0.1 U/g to about 1.8 U/g, on a dry-weight basis, where U is heparin-equivalent units.
The invention is directed, furthermore, to a coagulation control comprising human plasma and non-primate mammalian plasma, with the ratio of non-primate mammalian plasma to human plasma ranging from about 1:200 to about 1:5, and preferably from about 1:50 to about 1:20. When the coagulation control is a solution, the amount of non-primate mammalian plasma in the solution preferably ranges from about 0.5% to about 12%, by volume.
The invention is directed as well to methods for preparing such coagulation controls. According to one method, primate plasma, non-primate mammalian plasma and an anticoagulant such as a glycosaminoglycan and having activity as described above are combined to form a control solution having an anticoagulant concentration ranging from about 0.01 U/ml to about 0.15 U/ml, and preferably from about 0.01 U/ml to about 0.1 U/ml. Preferably, the human plasma and bovine plasma are combined with a heparinized buffer directly after thawing to form heparinized plasma solution that can be subsequently combined to form control solutions. According to another independent, but complementary method, a coagulation control composition is prepared by combining human plasma and non-primate mammalian plasma to form a control solution having non-primate mammalian plasma in an amount ranging from about 0.5% to about 12% by volume.
Preferred aspects of the invention additionally include methods for preparing a purified bovine plasma. In these methods, frozen bovine plasma is prepared or obtained (e.g. from commercial sources), thawed in an environment (e.g. a refrigerator) maintained a temperature ranging from about 2xc2x0 C. to about 8xc2x0 C. to allow particulates to form and come out of solution, and then treated to remove the particulates from the thawed bovine plasma. The freezing and cold-temperature thawing is preferably repeated at least once.
The invention is further directed to methods for evaluating a PT or APTT assay system. Such quality control methods generally include combining a PT reagent or an APTT reagent with a coagulation control to form an assay solution, detecting clot formation in the assay solution, and determining the time elapsed from formation of the assay solution to detection of clot formation in the assay solution. A comparison of the determined times can then be made with target times for the particular control being evaluated. For evaluation of a PT assay system, the coagulation control comprises a plasma and an anticoagulant having activity for enhancing the activity of antithrombin III (ATIII) and/or of heparin co-factor II (HCII) against thrombin and/or against blood clotting factors such as factors IXa, Xa and/or XIa. The anticoagulant is preferably a glycosaminoglycan and most preferably heparin, a heparin derivative or a heparin analog. For evaluation of either PT or APTT assay systems, the coagulation control comprises an abnormal plasma and anticoagulant such as a glycosaminoglycan having activity as described above. Alternatively, the coagulation control used for quality control of either PT or APTT assay systems comprises primate plasma, non-primate mammalian plasma and an anticoagulant such as a glycosaminoglycan having activity as described above.
The coagulation controls and methods of the present invention offer significant advantages over prior art control materials and methods. Controls disclosed and claimed herein have exceptional stability in reconstituted form, even when prepared in large-scale. commercial production facilities. The controls can be prepared from readily available materials, using conventional equipment, under FDA good manufacturing practices. As such, the coagulation controls of the invention offer a commercially attractive alternative to existing coagulation controls for use in connection with PT and APTT assays.
Other features, objects and advantages of the present invention will be in part apparent to those skilled in the art and in part pointed out hereinafter. All references cited in the instant specification are incorporated by reference. Moreover, as the patent and non-patent literature relating to the subject matter disclosed and/or claimed herein is substantial, many relevant references are available to a skilled artisan that will provide further instruction with respect to such subject matter.